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目的:探讨慢病毒介导的生长分化因子15(growth differentiation factor 15,GDF15)基因低表达对胶质瘤U373细胞对化疗药物替尼泊苷(teniposide,VM-26)和顺铂(cisplatin,DDP)耐药性的影响及其可能的机制。方法:将靶向GDF15的GDF15-RNAi克隆入慢病毒载体GV248,构建shRNA慢病毒LV-GDF15-RNAi,用无关序列构建阴性对照慢病毒LV-RNAi,分别稳定转染U373细胞,Western blotting检测转染对细胞内GDF15表达的影响。用梯度质量浓度的VM-26(0.1、0.5、2.5和12.5μg/ml)和DDP(0.08、0.4、2和10μg/ml)处理LV-GDF15-RNAi组和LV-RNAi组细胞48 h,MTT法、Hoechst/PI双染检测LVGDF15-RNAi转染对U373细胞VM-26、DDP耐药性的影响,Western blotting检测LV-GDF15-RNAi转染对U373细胞凋亡相关蛋白Bcl-2、Bcl-x L、P53和Caspase-3表达的影响。结果:成功构建稳定低表达GDF15的U373细胞系,LV-GDF15-RNAi组细胞内GDF15表达较LV-RNAi组和野生型U373细胞显著降低(0.013±0.001 vs 0.622±0.068、0.601±0.004,均P<0.01)。VM-26和DDP在最低浓度时,LV-GDF15-RNAi组细胞存活率即显著高于LV-RNAi组[VM-26 0.1μg/ml:(91.84±2.64)%vs(80.71±2.66)%,P<0.01;DDP 0.08μg/ml:(102.35±6.79)%vs(85.10±3.69)%,P<0.01],随药物浓度升高差异更加显著。相同浓度的VM-26或DDP处理下,LV-GDF15-RNAi组细胞凋亡数均少于LV-RNAi组;同时,LV-GDF15-RNAi组的Bcl-2和Bcl-x L的表达量比LV-RNAi组显著增多(P<0.05或P<0.01),Caspase-3和P53的表达量显著下降(P<0.05或P<0.01)。结论:下调胶质瘤U373细胞中GDF15水平能够增强细胞对VM-26和DDP的耐药性,其机制可能与GDF15调控Bcl-2、Bcl-x L、P53和Caspase-3的表达有关。
OBJECTIVE: To investigate the effect of lentivirus-mediated growth differentiation factor 15 (GDF15) gene silencing on glioma U373 cells on teniposide (VM-26) and cisplatin (DDP) ) Drug resistance and its possible mechanism. Methods: GDF15-RNAi targeting GDF15 was cloned into lentiviral vector GV248 to construct shRNA lentivirus LV-GDF15-RNAi. Negative control lentivirus LV-RNAi was constructed with unrelated sequence and transfected into U373 cells respectively. Effect of Dye on the Expression of GDF15 in Cells. The LV-GDF15-RNAi and LV-RNAi groups were treated with gradient concentrations of VM-26 (0.1,0.5,2.5 and 12.5μg / ml) and DDP (0.08,0.4,2 and 10μg / ml) The effect of LVGDF15-RNAi transfection on the drug resistance of U373 cells to VM-26 and DDP was detected by Hoechst / PI double staining. The expression of Bcl-2, Bcl- x L, P53 and Caspase-3 expression. Results: The expression of GDF15 in LV-GDF15-RNAi group was significantly lower than that in LV-RNAi group and wild-type U373 cells (U1383 cells, U1323 cells, P <0.01) <0.01). The survival rate of LV-GDF15-RNAi group was significantly higher than that of LV-RNAi group [VM-26 0.1μg / ml: (91.84 ± 2.64)% vs (80.71 ± 2.66)%, P <0.01; DDP 0.08μg / ml: (102.35 ± 6.79)% vs (85.10 ± 3.69)%, P <0.01]. The difference was more significant with the increase of drug concentration. At the same concentration of VM-26 or DDP treatment, the number of apoptotic cells in LV-GDF15-RNAi group was less than that in LV-RNAi group. Meanwhile, the expression of Bcl-2 and Bcl-xL in LV-GDF15- The expression of Caspase-3 and P53 in LV-RNAi group was significantly increased (P <0.05 or P <0.01) (P <0.05 or P <0.01). Conclusions: The down-regulation of GDF15 in glioma U373 cells can enhance the resistance of cells to VM-26 and DDP. The mechanism may be related to the regulation of Bcl-2, Bcl-xL, P53 and Caspase-3 by GDF15.